Deoxyribooligonucleoside methylphosphonates have a 3'-5' methylphosphonate linkage in place of the phosphodiester bond. These nucleic acid analogs are resistant to hydrolysis by nucleases and are taken up intact by mammalian cells in culture. The analogs form stable complexes with complementary sequences of single-strand nucleic acids and can be used to specifically probe and inhibit nucleic acid function both in vitro and in living cells. To fully exploit the use of oligonucleoside alkylphosphonates as specific inhibitors of cellular nucleic acids, efficient procedures for their syntheses and characterization are required. To this end we will explore new synthetic procedures including: (1) use of "activated" methylphosphonic acid derivatives as bifunctional phosphonylating/condensing agents, (2) use of methyldicholrophosphine and methyl-bis-azole phosphines as bifunctional phosphonylating/condensing agents, (3) use of arenesulfonic acid derivatives as coupling reagents and (4) use of silica gel and polystyrene polymer supports to assist in the rapid assembly and purification of the analogs. The mechanism(s) of the coupling reactions will be investigated by 31P and 13C nuclear magnetic resonance spectroscopy. This information will be used to design stereospecific coupling reagents. Procedures based on the Maxam/Gilbert chemical sequencing method will be developed to characterize the analogs. The configuration of the methylphosphonate group will be assigned based on the relative rates of enzymatic hydrolysis of adjacent phosphodiester linkages in specifically designed alternating methylphosphonate/ phosphodiester analogs. The synthetic procedures will be used to prepare a series of analogs whose base sequences are complementary to selected regions near the origin of replication of PhiX174 DNA and to the terminal redundant sequences of replication initiation site of Rous Sarcoma Virus RNA. The analogs will be tested in vitro for their abilities to prevent copying of the viral templates by T4-DNA polymerase or reverse transcriptase in a site specific manner. The effect of the analogs on viral replication in living cells will be examined.